Project Abstract Identification of the autoantigens that drive the pathogenic T cell response in type 1 diabetes (T1D) is a research goal of great importance because of the implications for biomarker discovery and disease prevention strategies. Our lab has focused on the role of CD4 T cells in both pathogenesis and regulation of disease with particular emphasis on their target antigens. Using a proteomic approach and the BDC panel of CD4 T cell clones as our antigen detection system, we recently discovered that the peptide ligands that activate several clones in our panel, including the prototype clone BDC-2.5, were formed through a novel post-translational modification involving formation of hybrid peptides between peptide cleavage products of ?-cell proteins such as ChgA or IAPP and sequences from insulin C-peptide. The discovery of hybrid insulin peptides (HIPs) as a new class of neoantigens raises questions as to the extent of HIP reactivity in T1D and whether T cells reactive to HIPs drive the autoimmune process. We hypothesize that HIP-reactive T cells can serve as biomarkers of disease and that the discovery of new HIP reactivities in NOD mice and patients with T1D will offer new possibilities to stage the disease before it occurs. To test this hypothesis, we propose to (1) investigate the T cell repertoire for hybrid insulin peptides (HIPs) in the NOD mouse; (2) discover C-peptide HIP reactivities using combinatorial peptide pools in T1D and at risk subjects and (3) monitor phenotype and TCR of HIP-reactive T cells over time in subjects at risk for T1D.